Eric Prina

The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages

Leishmania are protozoan parasites that, as amastigotes, live in the macrophages of mammalian hosts within compartments called parasitophorous vacuoles. These organelles share features with late endosomes/lysosomes and are also involved in the trafficking of several major histocompatibility complex (MHC)-encoded molecules. Improved knowledge of the parasitophorous vacuoles may help clarify how these protozoa persist in their hosts.

Real-Time PCR for Detection and Quantitation of Leishmania in Mouse Tissues

ABSTRACT Leishmania spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However, monitoring of the Leishmania burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly accurate real-time PCR assay with which to reproducibly detect and quantify the relative Leishmania major burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of L. major DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity over an at least 6-log DNA concentration range, corresponding to 0.1 to 104 parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same magnitude as that used for detection of L. donovani and L. amazonensis, but it was 100-fold less sensitive for L. mexicana. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher the unique properties of the life cycle of Leishmania spp .

Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function

ABSTRACT Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin − / − mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin − / − mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin − / − LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin − / − mice were not impaired in their capacity to process native OVA protein for I-A b -restricted presentation to CD4+ T lymphocytes or for H-2K b -restricted cross-presentation to CD8+ T lymphocytes. langerin − / − mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin − / − and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin − / − C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.

Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis

A LightCycler real-time polymerase chain reaction (PCR) assay has been developed to detect and differentiate four of the main Leishmania species of the Old World. The assay is based on fluorescence melting curve analysis of PCR products generated from the minicircles of kinetoplast DNA. According to the melting temperature, which is a function of GC/AT ratio, length and nucleotide sequences of the amplified product, Leishmania major was differentiated from L. donovani and from L. tropica and L. infantum. Melting curves analysis offers a rapid alternative for identification of species in diagnostic or epidemiological studies of leishmaniasis or asymptomatic parasitism.

Presentation of the Leishmania antigen LACK by infected macrophages is dependent upon the virulence of the phagocytosed parasites.

We have previously demonstrated that murine macrophages (Mϕ) infected with Leishmania promastigotes, in contrast to Mϕ infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I‐Ad‐restricted T cell hybrids and to the T cell clone 9.1‐2. These T cells react with the LACK (158 – 173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK‐specific T cell hybridoma OD12 by promastigote‐infected Mϕ is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log‐phase or stationary‐phase promastigotes of L. major or of L. amazonensis, Mϕ strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by Mϕ infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, Mϕ infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected Mϕ correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158 – 173)‐I‐Ad complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158 – 173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by Mϕ infected with log‐phase or stationary‐phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4+ T lymphocytes.

Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation.

In their mammalian hosts, Leishmania are obligate intracellular parasites that mainly reside in macrophages. They are also phagocytosed by dendritic cells (DCs), which play decisive roles in the induction and shaping of T cell-dependent immune responses. Little is known about the role of DCs in the Leishmania life cycle. Here, we examined the ability of mouse bone marrow-derived DCs to serve as hosts for L. amazonensis. Both infective stages of Leishmania (metacyclic promastigotes and amastigotes) could be phagocytosed by DCs, regardless of whether they had previously been experimentally opsonized with either the complement C3 component or specific antibodies. Parasites could survive and even multiply in these cells for at least 72 hours, within parasitophorous vacuoles displaying phagolysosomal characteristics and MHC class II and H-2M molecules. We then studied the degree of maturation reached by infected DCs according to the parasite stage internalised and the type of opsonin used. The cell surface expression of CD24, CD40, CD54, CD80, CD86, OX40L and MHC class II molecules was barely altered following infection with unopsonized promastigotes or amastigotes from nude mice or with C3-coated promastigotes. Even 69 hours post-phagocytosis, a large proportion of infected DCs remained phenotypically immature. In contrast, internalisation of antibody-opsonized promastigotes or amastigotes induced DCs to mature rapidly, as shown by the over-expression of costimulatory, adhesion and MHC class II molecules. Thus, in the absence of specific antibodies (e.g. shortly after infecting naive mammals), infected DCs may remain immature or semi-mature, meaning that they are unable to elicit an efficient anti-Leishmania T cell response. Absence of DC maturation or delayed/incomplete DC maturation could thus be beneficial for the parasites, allowing their establishment and amplification before the onset of immune responses.

Leishmania spp.: on the interactions they establish with antigen-presenting cells of their mammalian hosts

Identification of macrophages as host cells for the mammalian stage of Leishmania spp. traces back to about 40 years ago, but many questions concerning the ways these parasites establish themselves in these cells, which are endowed with potent innate microbicidal mechanisms, are still unanswered. It is known that microbicidal activities of macrophages can be enhanced or induced by effector T lymphocytes following the presentation of antigens via MHC class I or class II molecules expressed at the macrophage plasma membrane. However, Leishmania spp. have evolved mechanisms to evade or to interfere with antigen presentation processes, allowing parasites to partially resist these T cell-mediated immune responses. Recently, the presence of Leishmania amastigotes within dendritic cells has been reported suggesting that they could also be host cells for these parasites. Dendritic cells have been described as the only cells able to induce the activation of naive T lymphocytes. However, certain Leishmania species infect dendritic cells without inducing their maturation and impair the migration of these cells, which could delay the onset of the adaptive immune responses as both processes are required for naive T cell activation. This review examines how Leishmania spp. interact with these two cell types, macrophages and dendritic cells, and describes some of the strategies used by Leishmania spp. to survive in these inducible or constitutive antigen-presenting cells.

High Content Analysis of Primary Macrophages Hosting Proliferating Leishmania Amastigotes: Application to Anti-leishmanial Drug Discovery

Background/Objectives Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis. Methodology/Principal Findings We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline. Conclusions/Significance Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.

Virulence of Leishmania infantum Is Expressed as a Clonal and Dominant Phenotype in Experimental Infections

ABSTRACT Human Leishmania infantum infection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence in L. infantum visceral leishmaniasis is clonal and dominant in nature.

Kinetics of the intracellular differentiation of Leishmania amazonensis and internalization of host MHC molecules by the intermediate parasite stages

The establishment of Leishmania in mammals depends on the transformation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The appearance of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48 h but probable precursors of these organelles were detected at 12h p.i. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12-24 h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24 h. The data indicate that at 48 h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The appearance of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host MHC molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival.